Fluorescent polymeric particles have often found utility as markers and indicators in various biomedical assays. Fluorescent particles are usually obtained by embedding or diffusing a fluorescent dye according to the technique originally described by L. B. Bangs (Uniform Latex Particles; Seragen Diagnostics Inc. 1984, p. 40). Other methods are known in the art to stain particles with fluorescent dyes. The microparticles can then be analyzed manually or by other methods known in the art, but preferably using an automated technique, e.g., flow cytometry, such as disclosed in U.S. Pat. No. 4,665,024 issued to Mansour, et al.
The versatility of these particles can be enhanced by the incorporation in a single particle of a plurality of dyes, each dye having unique spectral characteristics. One skilled in the art would recognize that two or more dyes of varying proportions could be used to increase the permutation number of unique combinations of dyes on a single particle. While simple absorption of a single dye into a particle has proven adequate for most purposes, several problems arise when more than one dye is absorbed into a particle.
First, the close proximity of embedded dye molecules gives rise to significant amounts of fluorescent energy transfer. This energy transfer leads to fluorescent emissions that are inconsistent with relative dye concentrations and their original emission patterns.
A second problem arises when the dye substances used have differing solubilities in the solvent used to incorporate the dye in the particles. Since all dyes must be absorbed simultaneously, possible dye ratios are restricted by solvent properties.
A third problem that has been encountered when multiple dyes are embedded in microparticles is the change in dye spectra. Specifically, it has been noted that, when the particle is composed of crosslinked polystyrene, a significant broadening of the fluorescent emission peak occurs. This can result in an overlapping of the spectra of neighboring dyes.
One method that may circumvent these problems is to couple each dye substance to the surface of the particle chemically. This approach is, for example, disclosed in U.S. Pat. Nos. 5,194,300 to Cheung and 4,774,189 to Schwartz, whereby one or several fluorescent dyes are bound covalently to the surface of particles. This, however, leaves the dye molecule exposed to the environment, which can hasten decomposition by oxidation or other chemical attack. Additionally, a large number of surface binding sites would be occupied by dye and would be unavailable for the conjugation of analytical reactant molecules necessary to perform the assays.
Hence, it is desirable to have multicolored fluorescent particles, which avoid the above problems. This invention minimizes or eliminates these complications while maintaining the versatility of multi-dye particles.
Masson et al., disclose in U.S. Pat. No. 4,279,617, latex particles of relatively large diameter (e.g. 0.79 μm) coated with an analytical reactant, e.g., allergen, either by simple adsorption or by covalent coupling with cyanogen bromide or hydroxylated latex. A sample of human serum from a person suspected to have an allergic reaction is mixed with a suspension of these particles. The mixture is incubated and latex particles of a relatively smaller diameter (e.g. 0.08 μm) are then added. These smaller particles are coated with rabbit anti-IgE antibodies and if larger particles have IgE bound to the allergen these small particles will bind to these antibodies and will form by virtue of agglomeration reaction so-called agglutination particles, i.e., large particles surrounded by several smaller particles. However, these particles are bound to each other via noncovalent binding and the agglomeration occurs as the consequence and result of the presence of the analyte of interest. No admission is made that any reference cited in this specification is prior art. All references cited herein are hereby expressly incorporated by reference.